Indirubin (3, 2′-bi-indole) and its isoform, isoindigo (3, 3′-bi-indole) are the active antitumor components isolated from the Chinese medicinal herb, Qing-Dai or Indigofera tinctoria L. (Han, J. Traditional Chinese medicine and the search for new antineoplastic drugs. J. Ethnopharmacol. 1988; 24(1): 1-17).

Indirubin, isoindigo and numerous synthetic derivatives based on the bi-indole core structure have been shown to have very broad biological activities. Aza-indirubins and aza-isoindigos were synthesized by replacing a carbon or carbons with a nitrogen or nitrogens in the scaffolds of indirubin and isoindigo, respectively, and shown to have the biological activities similar to those of indirubins and isoindigos (Kritsanida, M. et al. Synthesis and antiproliferative activity of 7-azaindirubin-3′-oxime, a 7-aza isostere of the natural indirubin pharmacophore. J. Nat. Prod. 2009; 72(12): 2199-2202.; Wang, Z. et al. Synthesis and biological evaluation of 7-azaisoindigo derivatives. Arch Pharm (Weinheim) 2010; 343(3): 160-166.; Li X. et al. Anti-tumor effects in vitro and in vivo of novel 7-azaisoindigos. Chinese J. Cell Biol. 2013; 35(3): 334-340). The common feature of all these chemicals is that the two (aza)indoles are coupled to form structurally an extended pi-conjugated system. We believe that the similarity in three dimensional core structures of these compounds confers their similar biological functions, i.e., it is mainly the three dimensional structure, rather than just the chemical composition of the scaffold of these compounds, that is responsible for the biological activities. We hypothesized that modifying the chemical composition but maintaining such extended pi-conjugated system could result in new chemical entities with similar biological functions to those of indirubins and isoindigos. We, therefore, designed and synthesized two novel types of chemical entities, 1′-oxo-(aza)indirubins and 1′-oxo-(aza)isoindigos, by coupling (aza)indole with benzofuranone. Functional testing confirmed that, similar to indirubins and isoindigos, these novel molecules could also inhibit growth of tumor cells.
Natural products have always been a rich resource in drug discovery. Many important drugs, such as Aspirin, Digoxin, Paclitaxel, etc., were developed through understanding the biological activities and optimizing the chemical structures of natural products. A traditional Chinese medicinal receipe “Dang Gui Lu Hui Wan”, which contains the herb “Qing Dai” or indigo naturalis, had been used for a long time in China to treat chronic myeloid leukemia (CML). The active molecular components of the recipe had not been known until the 1960s when Chinese scientists identified indirubin (Wu, L., Yang, Y. & Zhu, Z. Studies on the active principles of indigofera tinctoria in the treatment of CML. Comm. Chinese Herb. Med. 1979; 9(1): 6-8). Subsequent research in China on the structure activity relationship of indirubins and their isomers, isoindigos, resulted in the development of the novel anti-cancer drugs, indirubin and meisoindigo (Meisoindigo, or N-methyl-isoindigo) (Institute of Haematology, Chinese Academy of Medical Sciences. Clinical studies of Dang Gui Lu Hui Wan in the treatment of CML. Chinese J. Intern. Med., 1979; 15: 86-88; Institute of Haematology, Chinese Academy of Medical Sciences. Clinical and experimental studies in the treatment of chronic granulocytic leukemia with indirubin. Zhonghua Nei Ke Za Zhi 1979; 18(2): 83-88; Wu, K., Zhang, M., Fang, Z. & Huang, L. Potential antileukemic agents, synthesis of derivatives of indirubin, indigo, and isoindigotin. Yao Xue Xue Bao 1985; 20(11): 821-826; Ji, X., Zhang, F., Liu, Y. & Gu, Q. Studies on the antineoplastic action of N-methylisoindigotin. Yao Xue Xue Bao. 1985; 20(4): 247-251). Indirubin and meisoindigo were the most effective anti-CML drugs in China before the targeted therapy drug, Imatinib Mesylate (Gleevec), was approved by the FDA in 2001. Due to different mechanisms of action (MOA), indirubin and meisoindigo can still be useful for Imatinib-resistant patients (Kim, W. et al. 5′-OH-5-nitro-Indirubin oxime (AGM130), an Indirubin derivative, induces apoptosis of Imatinib-resistant chronic myeloid leukemia cells. Leuk. Res. 2013; 37(4): 427-433).
The derivatives with a core structure similar to indirubin and isoindigo (hereafter collectively referred as “indirubins”) have exhibited a wide range of biological activities through modulating functions of various proteins, such as enzymes and transcriptional factors that are critical for cell proliferation and differentiation, apoptosis, inflammation, angiogenesis, metabolism, etc. Different derivatives can have different selectivity and potency (Duensing, S. et al. Cyclin-dependent kinase inhibitor indirubin-3′-oxime selectively inhibits human papillomavirus type 16 E7-induced numerical centrosome anomalies. Oncogene 2004; 23(50): 8206-8215; Lee, J. et al. Induction of apoptosis by a novel indirubin-5-nitro-3′-monoxime, a CDK inhibitor, in human lung cancer cells. Bioorg. Med. Chem. Lett. 2005; 15(17): 3948-3952; Moon, M. et al. Synthesis and structure-activity relationships of novel indirubin derivatives as potent anti-proliferative agents with CDK2 inhibitory activities. Bioorg. Med. Chem. 2006; 14(1): 237-246; Choi, S. et al. 5, 5′-substituted indirubin-3′-oxime derivatives as potent cyclin-dependent kinase inhibitors with anticancer activity. J. Med. Chem. 2010; 53(9): 3696-3706), and therefore can be effective in treating numerous different diseases by targeting various signaling pathways involved in the pathogeneses of the diseases.
Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases. The human genome contains 21 genes encoding CDKs and 5 other genes encoding CDK-like (CDKL) kinases (Malumbres, M. et al. Cyclin-dependent kinases: a family portrait. Nat. Cell Biol. 2009; 11(11): 1275-1276). Cyclins comprise a large family of proteins with diverse functions, each bound to its catalytic partner, CDK. By itself, CDKs exhibit little kinase activity. Cyclin bindings not only activate CDKs but also determine the substrates of the enzyme complexes. Sequential activation of CDKs and phosphorylation of key CDK substrates control each phase of cell cycle progression in all eukaryotes. Aberrant CDK activity leading to uncontrolled cell proliferation is a common feature of most cancer types (Sherr, C. Cancer cell cycles. Science 1996; 274(5293): 1672-1677). Targeting CDKs to arrest proliferation and induce apoptosis in neoplastic cells has been a promising strategy to treat cancer (Abate, A., Pentimalli, F., Esposito, L. & Giordano, A. ATP-noncompetitive CDK inhibitors for cancer therapy: an overview. Expert Opin. Investig. Drugs 2013; 22(7): 895-906). More than 10 small molecule CDK inhibitors with different chemical scaffolds have been tested in clinical trials over the past decade, however, none of them has been approved (Jorda, R., Paruch, K. & Krystof, V. Cyclin-dependent kinase inhibitors inspired by roscovitine: purine bioisosteres. Curr. Pharm. Des. 2012; 18(20): 2974-2980; Bose, P., Simmons, G. & Grant, S. Cyclin-dependent kinase inhibitor therapy for hematologic malignancies. Expert. Opin. Investig. Drugs 2013; 22(6): 723-738; Galons, H., Oumata, N., Gloulou, O. & Meijer, L. Cyclin-dependent kinase inhibitors closer to market launch?. Expert. Opin. Ther. Pat. 2013; 23(8): 945-963). One of the challenges that the CDK-targeted therapy faces is that the disruption of a CDK-mediated pathway has potentially serious consequences. Modest therapeutic effects are usually accompanied by severe side effects. The toxicity of a CDK inhibitor, depending on its potency, selectivity, and ATP-competitivity or non-competitivity, could be restricted in certain cell types or more general (Malumbres, M., Pevarello, P., Barbacid, M. & Bischoff, J. CDK inhibitors in cancer therapy: what is next?. Trends Pharmacol. Sci. 2008; 29(1): 16-21; Abate, A., Pentimalli, F., Esposito, L. & Giordano, A. ATP-noncompetitive CDK inhibitors for cancer therapy: an overview. Expert Opin. Investig. Drugs 2013; 22(7): 895-906).
Indirubin and its isoform derivative, meisoindigo, have shown antitumor activities with relatively low toxicities in treating leukemia in China. Indirubin acts as a potent CDK inhibitor by interacting with the ATP-binding site of CDK2 through van der Waals interactions and three hydrogen bonds (Hoesse, R. et al. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat. Cell Biol. 1999; 1(1): 60-67). An indirubin derivative, indirubin-3′-monoxime inhibits the proliferation of various types of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. Structural modifications can confer indirubin derivatives with different potencies and selectivities to different subtypes of CDKs. The unique properties of indirubins suggest their great therapeutic potential in the treatment of diseases with aberrant cell proliferation, such as various types of tumors (Eisenbrand, G., Hippe, F. & Jakobs, S. Molecular mechanisms of indirubin and its derivatives: novel anticancer molecules with their origin in traditional Chinese phytomedicine. J. Cancer Res. Clin. Oncol., 2004; 130: 627-635).
In addition to CDKs, the activities of other protein kinases, such as glycogen synthase kinase-3 (GSK3) can also be modulated by indirubins. GSK3 was first discovered for its ability to phosphorylate glycogen synthase (GS) and to consequently reduce GS activity. GSK3 is also able to phosphorylate other proteins in glucose metabolic pathways, such as signal transduction protein (insulin receptor substrate 1, IRS1), glycolytic enzyme (glucose 6-phosphatase, G6Pase) and gluconeogenic enzyme (phosphoenolpyruvate carboxykinase, PEPCK). Beyond the roles in glucose metabolism, GSK3 is involved in many signal transduction pathways related to innate as well as acquired or adaptive immune responses (Wang, H., Brown, J. & Martin, M. Glycogen synthase kinase 3: a point of convergence for the host inflammatory response. Cytokine 2011; 53(2): 130-140). GSK3 is also one of the key enzymes in neuronal development and regeneration (Seira, O. & Del Rio, J. Glycogen Synthase Kinase 3 Beta (GSK3β) at the Tip of Neuronal Development and Regeneration. Mol. Neurobiol. Mol. Neurobiol. 2014; 49(2): 931-44). Aberrant activity of GSK3 is correlated with degeneration of neuronal cells and the deposit of amyloid β (Aβ) in the brain and is able to directly accelerate the process of Aβ formation and tau protein over-phosphorylation, which is the cause of neurofibrillary tangles. Moreover, inhibition of GSK3 activity was found to be a mechanism through which certain antipsychotic drugs work (Jope, R., Yuskaitis, C. & Beurel, E. Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics. Neurochem Res. 2007; 32(4-5): 577-595). Targeting GSK3, therefore, can have very broad therapeutic applications (Maes, M. et al. New drug targets in depression: inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors. Inflammopharmacology 2012; 20(3): 127-150). It can be used to treat disorders of glucose metabolism (Gao, C., Hölscher, C., Liu, Y. & Li, L. GSK3: a key target for the development of novel treatments for type 2 diabetes mellitus and Alzheimer disease. Rev. Neurosci. 2011; 23(1): 1-11), such as type 2 diabetes (T2D); inflammatory and autoimmune diseases (Beurel, E., Michalek, S. & Jope, R. Innate and adaptive immune responses regulated by glycogen synthase kinase-3 (GSK3). Trends Immunol. 2010; 31(1): 24-31), such as arthritis; neurodegenerations (Ma, T. GSK3 in Alzheimer's Disease: Mind the Isoforms. J. Alzheimers Dis. 2014; 39(4): 707-710), such as Alzheimer's; and psychoses (Cole, A. Glycogen synthase kinase 3 substrates in mood disorders and schizophrenia. FEBS J. 2013; 280(21): 5213-5227), such as schizophrenia, etc. Indirubin is the first type of compound shown to inhibit GSK3 activities at lower nanomolar concentrations (Leclerc, S. et al. Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?. J. Biol. Chem. 2001; 276(1): 251-256). Indirubins can, therefore, be used to treat various types of diseases as described above.
Not limited to protein kinases, indirubins are also able to modulate activities of other key components in signal transduction pathways, and therefore affect cell proliferation and differentiation at different stages. Signal transducer and activator of transcription (STAT) is a family of transcriptional factors important in many cellular processes, and plays key roles in immune response, host defense, hematopoiesis, angiogenesis, metabolism, oncogenesis, etc. (O'Shea, J., Holland, S. & Staudt, L. JAKs and STATs in immunity, immunodeficiency, and cancer. N. Engl. J. Med. 2013; 368(2): 161-170). Janus kinase-STAT (JAK-STAT), originally identified as the signaling cascade downstream of cytokine receptors, provides a direct pathway to translate extracellular signals from a variety of ligands including cytokines, hormones and growth factors, etc., into a transcriptional response. Activated STATs binding to specific regulatory DNA sequences activate or repress the transcription of their target genes. Among the 7 members of the STAT family, STAT3 is indispensable in the differentiation and the activation of Th17, a type of T-helper cell critically involved in autoimmune diseases and tumors (Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 2009; 326(5955): 986-991; Camporeale, A. & Poli, V. IL-6, IL-17 and STAT3: a holy trinity in auto-immunity?. Front Biosci (Landmark Ed) 2012; 17: 2306-2326). Imbalance of Th17 and regulatory T cells (Treg) is the major cause of many inflammatory and autoimmune disorders. Targeting STAT3 therefore holds great promise for developing drugs with very broad indications (Mankan, A. & Greten, F. Inhibiting signal transducer and activator of transcription 3: rationality and rationale design of inhibitors. Expert Opin. Investig. Drugs 2011; 20(9): 1263-1275). Potent and selective STAT3 inhibitors can be effective therapeutics not only for different tumors (Page, B., Ball, D. & Gunning, P. Signal transducer and activator of transcription 3 inhibitors: a patent review. Expert. Opin. Ther. Pat. 2011; 21(1): 65-83.; Wang, X., Crowe, P., Goldstein, D. & Yang, J. STAT3 inhibition, a novel approach to enhancing targeted therapy in human cancers (review). Int. J. Oncol. 2012; 41(4): 1181-1191), but also for various other inflammatory or immune-related diseases in which STAT3 or Th17 is involved (Ghoreschi, K. et al. T helper 17 cell heterogeneity and pathogenicity in autoimmune disease. Trends Immunol. 2011; 32(9): 395-401), such as rheumatoid arthritis (Ju, J. et al. Modulation of STAT-3 in rheumatoid synovial T cells suppresses Th17 differentiation and increases the proportion of Treg cells. Arthritis Rheum. 2012; 64(11): 3543-3552), inflammatory bowel diseases (IBD) (Li, Y., de Haar, C., Peppelenbosch, M. & van der Woude, C. New insights into the role of STAT3 in IBD. Inflamm Bowel Dis. 2012; 18(6): 1177-1183), dermatitis and psoriasis (Kim, M. et al. Indirubin, a purple 3,2-bisindole, inhibited allergic contact dermatitis via regulating T helper (Th)-mediated immune system in DNCB-induced model. J. Ethnopharmacol. 2013; 145(1): 214-219.; Novelli, L., Chimenti, M., Chiricozzi, A. & Perricone, R. The new era for the treatment of psoriasis and psoriatic arthritis: perspectives and validated strategies. Autoimmun Rev. 2014; 13(1): 64-69), systemic lupus erythematosus (SLE) (Nalbandian, A., Crispin, J. & Tsokos, G. Interleukin-17 and systemic lupus erythematosus: current concepts. Clin. Exp. Immunol. 2009; 157(2): 209-215), type 1 diabetes (T1D) (Shao, S. et al. Th17 cells in type 1 diabetes. Cell Immunol. 2012; 280(1): 16-21), restenosis (Schwaiberger, A. et al. Indirubin-3′-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo. Arterioscler. Thromb. Vasc. Biol. 2010; 30(12): 2475-2481), etc.
Indirubins can inhibit STAT3 signaling (Nam, S. et al. Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in human cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2005; 102(17): 5998-6003), prevent STAT3 from being activated (Aggarwal, B. et al. Targeting signal-transducer-and-activator-of-transcription-3 for prevention and therapy of cancer: modern target but ancient solution. Ann. N. Y. Acad. Sci. 2006; 1091: 151-169), block tumor angiogenesis (Zhang, X. et al. Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2-mediated JAK/STAT3 signaling in endothelial cell. Int. J. Cancer 2011; 129(10): 2502-2511.), induce cancer cell apoptosis (Ribas, J. et al. 7-Bromoindirubin-3′-oxime induces caspase-independent cell death. Oncogene 2006; 25(47): 6304-6318), inhibit Th17 cell differentiation (Glatigny S, et al. Treatment of collagen-induced arthritis by Natura-alpha via regulation of Th-1/Th-17 responses. Eur. J. Immunol. 2010. 40(2): 460-469), etc. Both the mechanism of action and the experimental evidence indicate that indirubins can be utilized to treat a number of diseases as described above, including but not limited to, different types of tumors and various inflammatory and autoimmune diseases.
The broad biological activities of indirubins have also been observed through their regulatory effects on multiple signaling transduction pathways, numerous cytokines and different types of cells. Indirubins can induce neutrophil production while suppressing Th17 cell differentiation (Suzuki, K. et al. Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differentiation of human myelocytic leukaemia HL-60 cells. Br. J. Haematol. 2005; 130(5): 681-190); inhibit the maturation of monocyte-derived dendritic cells (Vlachos, C. et al. Malassezia-derived indoles activate the aryl hydrocarbon receptor and inhibit Toll-like receptor-induced maturation in monocyte-derived dendritic cells. Br. J. Dermatol. 2012; 167(3): 496-505.); modulate the functions of macrophages (Babcock, A., Anderson, A. & Rice, C. Indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804) is a potent modulator of LPS-stimulated macrophage functions. Toxicol. Appl. Pharmacol. 2013; 266(1): 157-166); block smooth muscle cell proliferation (Schwaiberger, A. et al. Indirubin-3′-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo. Arterioscler. Thromb. Vasc. Biol. 2010; 30(12): 2475-2481); increase neurogenesis of human neural progenitor cells (Lange, C. et al. Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells. Neurosci. Lett. 2011; 488(1): 36-40.; Castelo-Branco, G., Rawal, N. & Arenas, E. GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J. Cell Sci. 2004; 117(Pt 24): 5731-5737); decrease adipocyte cell differentiation (Choi, O. et al. The small molecule indirubin-3′-oxime activates Wnt/β-catenin signaling and inhibits adipocyte differentiation and obesity. Int. J. Obes (Lond). 2013; 209: 1-9); impair mitochondria functions (Varela, A. et al. Indirubin-3′-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction. Toxicol. Appl. Pharmacol. 2008; 233(2): 179-185); inhibit production of inflammatory cytokines (Kunikata, T. et al. Indirubin inhibits inflammatory reactions in delayed-type hypersensitivity. Eur. J. Pharmacol. 2000; 410(1): 93-100.; Glatigny S, et al. Treatment of collagen-induced arthritis by Natura-alpha via regulation of Th-1/Th-17 responses. Eur. J. Immunol. 2010. 40(2): 460-469); etc. In addition to the JAK/STAT pathway, other signaling transduction pathways on which indirubins have shown effects include the NFκB and JNK pathways (Kim, J. & Park, G. Indirubin-3-monoxime exhibits anti-inflammatory properties by down-regulating NF-κB and JNK signaling pathways in lipopolysaccharide-treated RAW264.7 cells. Inflamm. Res. 2012; 61(4): 319-325); the Wnt/β-Catenin pathway (Zahoor, M., Cha, P., Min, D. & Choi, K. Indirubin-3′-Oxime Reverses Bone Loss in Ovariectomized, Hindlimb-Unloaded Mice via Activation of the Wnt/β-Catenin Signaling. J. Bone Miner Res. 2014; 29(5): 1196-1205); the aryl hydrocarbon receptor (AHR) pathway (Stevens, E., Mezrich, J. & Bradfield, C. The aryl hydrocarbon receptor: a perspective on potential roles in the immune system. Immunology 2009; 127(3): 299-311); etc. Different indirubin derivatives with different selectivities, therefore, can further be used for the treatments of diseases with different etiopathogeneses, such as cardiovascular diseases (Schwaiberger, A. et al. Indirubin-3′-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo. Arterioscler. Thromb. Vasc. Biol. 2010; 30(12): 2475-2481), obesity (Choi, O. et al. The small molecule indirubin-3′-oxime activates Wnt/β-catenin signaling and inhibits adipocyte differentiation and obesity. Int. J. Obes (Lond). 2013; 209: 1-9), osteoporosis (Zahoor, M., Cha, P., Min, D. & Choi, K. Indirubin-3′-Oxime Reverses Bone Loss in Ovariectomized, Hindlimb-Unloaded Mice via Activation of the Wnt/β-Catenin Signaling. J. Bone Miner Res. 2014; 29(5): 1196-1205), asthma (Gupta, S., Sundaram, C., Reuter, S. & Aggarwal, B. Inhibiting NF-κB activation by small molecules as a therapeutic strategy. Biochim Biophys Acta 2010; 1799 (10-12): 775-787.; Mak, N K. et al. Inhibition of RANTES expression by indirubin in influenza virus-infected human bronchial epithelial cells. Biochem Pharmacol. 2004; 67(1): 167-74), aging (Spindler, S. et al. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using drosophila. 2012; PLoS One, 7(2): e29782), graft-vs-host disease (GVHD) (Stevens, E., Mezrich, J. & Bradfield, C. The aryl hydrocarbon receptor: a perspective on potential roles in the immune system. Immunology 2009; 127(3): 299-311), and viral infections including encephalitis, AIDS, etc. (Chang, S. et al. Antiviral activity of isatis indigotica extract and its derived indirubin against Japanese encephalitis virus. Evid Based Complement Alternat Med. 2012; 2012(925830): 1-7; Heredia, A. et al. Indirubin 3′-onoxime, from a Chinese traditional herbal formula, suppresses viremia in humanized mice infected with multidrug-resistant HIV. AIDS Res. Hum. Retroviruses 2014; 30(5): 403-406).
As described above, indirubins can inhibit activation of different kinases and various signaling transduction pathways that are critically involved in the pathogeneses of numerous diseases. Their broad activities and mild toxicities make indirubins promising candidates for drug development. Their poor solubility in water and in lipid, however, has been one of the hurdles that have prevented them from being successful drugs. Many efforts have been devoted to modifying indirubins for better potency, specificity and bioavailability, but with only limited success. It is, therefore, necessary to develop new types of chemical entities with biological activities similar to those of indirubins, but with more desirable druggable properties.